Normal human cells in vitro exhibit a stringent limitation of division capacity in contract to tumor-derived and virus-, carcinogen- or irradiation-transformed cells that can divide indefinitely (immortal). We do not yet understand the mechanisms that limit the division potential of normal human cells or the changes occurring to yield immortal cells. What we have found is that the immortal phenotype results from recessive changes in normal cell growth control and that there are multiple ways by which cells can become immortal. In this study we propose to systematically separate a variety of immortal human cell lines into complementation groups which, when fused with each other, yield hybrids having limited division potential. The number of complementation groups into which immortal cells can be assigned will give an estimation of the number of control processes present in the normal cell that can be modified to result in uncontrolled cell division. The experiments are also designed to demonstrate the effect, if any, of cell derivation, type of tumor, inducer of immortality and presence of active oncogenes on the assignment of complementation group. In addition, they will demonstrate changes in cellular proto-oncongene expression as hybrids divide and cease division and in the variant immortal cells that arise in non-dividing hybrid populations. The results should be of intrinsic interest to investigators studying various aspects of growth control. Furthermore, they will allow us, in the future, to take a directed approach to the study of the molecular basis of immortalization. Cell lines representative of each complementation group can be analyzed to determine what recessive changes have occurred in normal cell growth control to result in immortality in each case, rather than study randomly selected immortal cell lines.